Characterization and Identification of a Novel Candidate Vaccine Protein through Systematic Analysis of Extracellular Proteins of Erysipelothrix rhusiopathiae

Abstract

dErysipelothrix rhusiopathiae, the causative agent of swine erysipelas, is a facultative intracellular Gram-positive bacterium. It has been shown that animals immunized with a filtrate from E. rhusiopathiae cultures are protected against lethal challenge. In this study, we identified and characterized the extracellular proteins of E. rhusiopathiae to search for novel vaccine antigens. A concentrated culture supernatant from the E. rhusiopathiae Fujisawa strain, which has been found to induce protection in mice, was analyzed using two-dimensional electrophoresis. From more than 40 confirmed protein spots, 16 major protein spots were selected and subjected to N-terminal amino acid sequence determination, and 14 protein spots were successfully identified. The identified proteins included housekeeping proteins and other metabolic enzymes. We searched for surface-localized proteins by analyzing the genomes of two E. rhusiopathiae strains: Fujisawa and ATCC 19414. Genome analysis revealed that the ATCC 19414 strain has three putative surface-exposed choline-binding proteins (CBPs): CbpA, CbpB, and CbpC. Each CBP contains a putative choline-binding domain. The CbpC gene is mutated in Fujisawa, becoming a nonfunctional pseudogene. Immunogold electron microscopy confirmed that CbpA and CbpB, as well as the majority of the metabolic enzymes examined, are associated with the cell surface of E. rhusiopathiae Fujisawa. Immunization with recombinant CbpB, but not with other recombinant CBPs or metabolic enzymes, protected mice against lethal challenge. A phagocytosis assay revealed that antiserum against CbpB promoted opsonin-mediated phagocytosis by murine macrophages in vitro. The protective capabilities of CbpB were confirmed in pigs, suggesting that CbpB could be used as a vaccine antigen.

Identification of the Chromosomal Region Essential for Serovar-Specific Antigen and Virulence of Serovar 1 and 2 Strains of Erysipelothrix rhusiopathiae

“…The Escherichia coli strains used were JM109 (Toyobo, Tokyo, Japan) and DH5␣ (Toyobo). Cultivation of the E. coli strains was performed as described previously (23).…”

“…In the genus Erysipelothrix strains, heat-stable peptidoglycan antigens are used for serological classification using a double agar-gel precipitation test with type-specific rabbit antisera (12). Serovars are closely related to clinical forms; among the serovars assigned to the species E. rhusiopathiae (serovars 1a, 1b, 2,4,5,6,8,9,11,12,15,16,17,19,21,23, and N; serovar N lacks serovar-specific antigens), serovars 1a, 1b, and 2 are frequently isolated from diseased pigs (13)(14)(15)(16)(17)(18). However, it has not been clarified why these serovar strains, especially serovar 1a strains, are pathogenic to pigs, and the genetic and antigenic relationship between these serovars and serovar N has not been clarified (11).…”

Genomic and Immunogenic Protein Diversity of Erysipelothrix rhusiopathiae Isolated From Pigs in Great Britain: Implications for Vaccine Protection

“…The impact of these differences -if any -would be valuable to explore. The most conserved surface protein -GAPDH -has been confirmed to be expressed at the cell surface (Shi et al, 2013), and like SpaA, RspA and RspB, has a role in endothelial adhesion (Zhu et al, 2017c). It was recently found to provide good protection in both murine and porcine challenge models (Zhu et al, 2018).…”

“…Additional surface proteins that have a demonstrated role in immunogenicity of E. rhusiopathiae include rhusiopathiae surface protein A (RspA) (Shimoji et al, 2003), choline binding protein B (cbpB) (Shi et al, 2013;Zhu et al, 2018) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Zhu et al, 2018); these proteins have all elicited protection during challenge studies in mice, and the latter two also in pigs. An additional eight putative surface proteins were recently found to give rise to varying degrees of protective immunity in mice (Hu et al, 2017; Table 1).…”

Erysipelothrix Spp.: Past, Present, and Future Directions in Vaccine Research

“…Other E. rhusiopathiae protective antigens reported so far include RspA (rhusiopathiae surface protein A) (130), CbpB (choline-binding protein) (131,132), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (133). These protective antigens play important roles in biofilm formation (RspA) (130) and adhesion to porcine endothelial cells (SpaA and GAPDH) (133,134), and to extracellular matrix proteins, including fibronectin (RspA and GAPDH) (130,133), collagens (RspA) (130), and plasminogen (GAPDH) (133).…”